Abstract Objective: Determine whether LDH-A knockdown enhances CAR T-cell tumor-targeting and treatment response.Background: It has been shown that T-cells are restricted from entering many human and murine solid tumors, including both murine and human prostate tumors. CD3(+) T-cells are restricted from Myc-CaP murine prostate tumors and localize around the periphery of the tumor nodules (1). Human prostate specific membrane antigen (hPSMA) targeted CAR T-cells are also restricted from hPSMA(+) Myc-CaP tumors and this is only partially reversed by anti-PD1 treatment (1). This report describes the benefit of LDH-A depletion and a greater response of CAR T-cells targeting Myc-CaP tumors with low expression of LDH-A. Methods: LDH-A depletion in hPSMA(+) Myc-CaP cells bearing a bioluminescence reporter (Renilla Luciferase), was achieved by shRNA knockdown (KD) (1). A scrambled IgG shRNA was used as a control (NC). LDH-A expression was quantified by digital droplet PCR (ddPCR), Western blotting and LDH enzyme activity. The glycolytic activity of KD and NC cells was measured using Seahorse XF96 and XFp analyzers. Intratumoral lactate levels were monitored by magnetic resonance spectroscopy (MRS). The preparation and characterization of “second generation” hPSMA-directed CAR T-cells and the hPSMA(+) Myc-CaP tumor models in SCID mice have been described (1). To monitor CAR T-cell trafficking, T-cells were transduced with a Click Beetle Red luciferase reporter to enable efficient visualization by bioluminescence imaging (BLI) of CAR T-cell trafficking, persistence and viability within the hPSMA(+)Myc-CaP tumor mass. Tumor volume was calculated from caliper measurements. CD31 and PD-L1 expression was quantified with immunofluorescent staining and Metamorph Offline image analysis. Results and Discussion: These studies demonstrated that LDH-A KD was the dominant factor in reducing tumor growth. The difference in tumor doubling time (DT) between KD and NC tumors in the presence of CAR T-cell therapy was significant (p<0.0001): DT= 5.0±0.3 vs 2.9±0.5 days, respectively. A significant difference was also found in the absence of CAR T-cell therapy. Although LDH-A KD produced the larger single-treatment effect, the addition of CAR T-cells was additive. To explain these observations and study the effect of LDH-A KD on tumor metabolism and microenvironment, Seahorse and MRS experiments were performed. LDH-A depletion (KD) resulted in: lower rates of basal (NC: 24.0±1.9 vs KD: 6.9±1.9 pmol H+/min/µg protein; p<0.0001) and compensatory glycolysis (NC: 43.0±2.3 vs KD: 18.3±3.9 pmol H+/min/µg protein, p<0.0001), and lactate production. In contrast, basal and maximal respiration increased following LDH-A KD (NC: 4.0±0.8 vs KD: 8.3±0.6, p=0.0011, and NC: 6.7±1.4 vs KD: 13.8±0.6 pmol O2/min/µg protein, p=0.0003, respectively). However, total tumor lactate, measured in vivo by MRS, showed only marginal differences between KD and NC tumors. Nevertheless, there were significant differences in CAR T-cell trafficking-to and persistence-in KD compared to NC tumors, and the increase in tumor doubling time was directly related to the CAR T-cell expansion ratio. Interestingly, tumor vascularity (CD31), but not PD-L1, was significantly lower in KD compared to NC tumors. Necrosis was minimal in both KD and NC tumors, with or without CAR T-cell treatment. Conclusions: LDHA knockdown (KD) in the Myc-CaP murine prostate tumor model has a significant effect on tumor cell metabolism resulting in reduced glycolysis, reduced lactate production and increased oxidative phosphorylation that impacts the tumor microenvironment. The LDH-A KD induced changes in tumor metabolism and the microenvironment diminish Myc-CaP tumor growth rate and impact CAR T-cell trafficking and persistence in the tumor. Future studies will compare the effects of anti-PD1 and anti-CTLA4 therapy in combination with LDH-A knockdown. Reference: 1. Serganova I, et al. Molecular Therapy: Oncolytics 2017;4:41. Citation Format: Mayuresh M. Mane, Khalid Shalaby, Ivan Cohen, Avi Albeg, Jenny Ijoma, Myat Ko, Masatomo Maeda, Kiranmayi Vemuri, Jaya Satagopan, Anna Moroz, Juan Zurita, Larissa Shenker, Ellen Ackerstaff, Masahiro Shindo, Ekaterina Moroz, Maxim A. Moroz, Inna Serganova, Jason Koutcher, Vladimir Ponomarev, Ronald G. Blasberg. The effect of lactate dehydrogenase-A (LDH-A) knockdown and human prostate-specific membrane antigen (hPSMA) directed CAR T-cell treatment on hPSMA(+) Myc-CaP tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A089.
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Nov 13, 2019
Tobias Dittrich + 7
